Research Article: ‘Relax and Repair’ to restrain aging

Date Published: October 30, 2011

Publisher: Impact Journals LLC

Author(s): Vaidehi Krishnan, Baohua Liu, Zhongjun Zhou.

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Abstract

The maintenance of genomic integrity requires the precise identification and repair of DNA damage. Since DNA is packaged and condensed into higher order chromatin, the events associated with DNA damage recognition and repair are orchestrated within the layers of chromatin. Very similar to transcription, during DNA repair, chromatin remodelling events and histone modifications act in concert to ‘open’ and relax chromatin structure so that repair proteins can gain access to DNA damage sites. One such histone mark critical for maintaining chromatin structure is acetylated lysine 16 of histone H4 (AcH4K16), a modification that can disrupt higher order chromatin organization and convert it into a more ‘relaxed’ configuration. We have recently shown that impaired H4K16 acetylation delays the accumulation of repair proteins to double strand break (DSB) sites which results in defective genome maintenance and accelerated aging in a laminopathy-based premature aging mouse model. These results support the idea that epigenetic factors may directly contribute to genomic instability and aging by regulating the efficiency of DSB repair. In this article, the interplay between epigenetic misregulation, defective DNA repair and aging is discussed.

Partial Text

The genomes of organisms are organized in the form of a fundamental structure called chromatin in which the repeating nucleosomes form the basic unit. The nucleosome consists of 147 bp of DNA wound 1.7 times around an octamer composed of the four core histones, H2A, H2B, H3 and H4. Multiple nucleosomes are further linked by DNA stretches that are occupied by linker histone H1, to form the 10-nm fibre or ‘beads on a string’ type of arrangement. Chromatin fibres undergo compaction through intramolecular nucleosome-nucleosome interactions to form the 30 nM chromatin fibres. At the next level of organization, chromatin is further stacked and folded to give rise to 100-400 nm interphase chromatin fibres. The DNA that is eventually folded into chromosomes has already undergone compaction by about 10,000 fold. The packaging of DNA into condensed and often inaccessible chromatin imposes a significant constraint for the efficient repair of DNA double strand breaks (DSBs). Recent efforts have been directed towards understanding how the DNA repair proteins gain access to and repair, when DNA damage is embedded within chromatin fibres.

Cells utilize two distinct mechanisms to modulate chromatin dynamics during DNA repair. These mechanisms include the post translational modifications of histones and ATP-dependent chromatin remodelling. According to the ‘histone code hypothesis’, the biological outcome of histone modifications is manifested by providing a signalling platform for the recruitment of downstream effector and reader protein or by the physical modulation of chromatin structure [1]. Accordingly, histones within chromosomes are subjected to several forms of post translational modifications such as phosphorylation, ubiquitination, methylation, and acetylation and these modifications can either create or eliminate binding sites for non-histone proteins that mediate DNA repair or modify chromatin structure [2]. The earliest identified and one of the most important histone modifications during DNA repair is the phosphorylation of histone H2AX at the C-terminal residue corresponding to Ser139 (γ-H2AX) by the key DNA damage-responsive kinase, ATM [3].During DSB repair, phosphorylated H2AX forms a specialised chromatin compartment capable of recruiting and retaining DNA repair factors [4]. H2AX phosphorylation spreads over a 2 Mb domain on each side of the DSB, and acts as a docking site for several DNA repair proteins such as the mediator, MDC1. Thus, H2AX phosphorylation acts as an important cue for the stable retention of DNA repair proteins which form microscopically discernible foci, called as irradiation-induced foci (IRIF) [5]. It is now recognized that apart from phosphorylation, H2AX is monoubiquitinated and later di and poly-ubiquitinated in a DNA damage-dependent manner. According to current understanding, several ubiquitin ligases including RNF8, RNF168, RNF2, Bmi1 and Herc2 are responsible for the completion ubiquitination of γ-H2AX [6]. In turn, ubiquitinated histones promote the recruitment of DNA repair proteins, Brca1and 53BP1 which directly participate in the repair of DSBs by homologous recombination (HR) or non-homologous end joining mechanisms (NHEJ). Apart from histone ubiquitination, histone methylation also plays an important role in DSB repair processes. Trimethylated H3K9 is known to be an important component of heterochromatin and the heterochromatin 1 proteins, HP1 α,β,δ, bind to trimethylated H3K9, contributing to heterochromatin maintenance. Upon DSB induction, HP1 β is phosphorylated in a casein kinase-dependent manner, which promotes HP1 β dissociation and γ -H2AX phosphorylation [7]. Following HP1 β dissociation, the MYST family histone acetyltransferase (HAT) Tip60, is recruited to trimethylated H3K9 resulting in the stimulation of its HAT activity [8]. In other examples, constitutive dimethylation of histone H3K79 by Dot1L and DNA damage-inducible H4K20 dimethylation by MMSET also provide a recruitment platform for the DSB repair protein, 53BP1 [9, 10]. Thus, in general, histone methylation mainly provides binding sites for the direct recruitment for downstream repair proteins.

Amongst the most prominent phenotypes associated with defective DSB repair in both humans and mice, is the onset of accelerated aging (progeria). In several correlative studies, γ -H2AX foci containing senescent cells increase with age in humans, mice and primates, leading to the model that inefficient DSB processing and repair can activate cellular senescence pathways and initiate premature aging [29-31]. This notion is strengthened with the observation that knockout-mice defective for DSB repair often show an accelerated aging phenotype and conversely, defective DNA repair is a common phenotype in human patients suffering from premature aging (progeria) syndromes [32-33].

Lamin A is a structural constituent of a subnuclear compartment called as the nuclear matrix [47]. The nuclear matrix is a nuclear subcellular compartment that is thought to provide a scaffold to facilitate chromatin organization and transcriptional regulation [48]. The nuclear matrix is recognized as a distinct subcellular compartment constituted of detergent and DNase-insoluble proteins.The observation that lamin A mutants are defective for DNA repair was surprising given that there was no precedent of a nuclear matrix-associated structural protein capable of regulating DNA repair.

How do reduced global H4K16 acetylation levels affect chromatin structure? In elegant work by Shogren-Knaak et al., the incorporation of acetylated K16 of H4 into nucleosomal arrays was demonstrated to impede the ability of chromatin to form cross-fibre interactions and this converted chromatin into an ‘open’ conformation [50]. In crystallization studies, it was observed that N- terminal of H4 tail of one mononucleosome interacted with the H2A/H2B of an adjacent mononucleosome, suggesting that this interaction might mediate chromatin compaction. Using chemical ligation, histone H4 that uniformly acetylated only at lysine 16 was prepared and intramolecular chromatin compaction was studied. Strikingly, these studies revealed that upon H4K16 acetylation, chromatin fibres could not achieve the fully compacted 30 nm fibre state adopted by unacetylated arrays.

A simplified view of the above model is that promoting chromatin accessibility by increasing histone acetylation levels can improve DNA repair by increasing the recruitment of DNA repair proteins. HDAC inhibitors prove useful for this purpose, since they are well-established as anti-cancer agents and several pharmacological agents are validated to increase histone acetylation levels [54]. Based on the rationale that HDAC inhibitors can promote DNA damage recognition and repair by promoting global chromatin relaxation, it was tested whether cellular senescence and premature aging phenotypes may be attenuated in Zmpste24-null cells upon HDAC inhibition. Interestingly, when Zmpste24-null cells were treated with HDAC inhibitors, sodium butyrate or trichostatin A, a significant reduction in the accumulation of unrepaired DSBs and an overall improvement in cell survival after DNA damage were noticed. Importantly, when Zmpste24-null mice were fed with sodium butyrate, a modest extension of life span and amelioration of premature aging phenotypes was observed, suggesting that HDAC inhibition might have a therapeutic potential. It is certainly possible that HDAC inhibitors may be non-specific in their action and thus it might be difficult to establish whether the extension of longevity is due to restoration of histone acetylation levels or due to other indirect benefits. Secondly, the dosage of HDAC inhibitors have to be carefully titrated since an excessive concentration might lead to toxicity. Despite these caveats, there are now several studies reporting the beneficial effects of HDAC inhibitors on aging. Recently, HDAC inhibitors were shown to improve DNA repair in an oncogene-induced senescence model by causing chromatin relaxation [55]. In other studies, HDAC inhibitors have been shown to increase learning ability, delay age-dependent neurodegeneration, delay Alzheimer’s disease progression in mouse models, accelerate age-associated osteogenesis, and increase life span of worms in a dietary restriction model [56-60]. It still needs to be established if the therapeutic benefits of HDAC inhibitors in these model systems are linked to their ability to directly promote DNA repair by the relaxation of chromatin structure.

Zmpste24-null mice or human HGPS cells have been considered as segmental progeroid syndromes, in that they only partially recapitulate the phenotypes associated with normal aging. Hence, it was believed that experiments conducted on premature aging syndromes may not accurately reflect physiological aging. This view is gradually changing owing to efforts made to detect progerin or prelamin A accumulation during normal aging. In the first such report, rare fibroblasts cultured from elderly individuals were found to exhibit nuclear abnormalities similar to HGPS cells [61]. Progerin transcripts and progerin protein could be detected from cells obtained from healthy individuals and more importantly the specific knockdown of progerin rescued age-associated nuclear deformities. In another study, the technical difficulty associated with progerin detection was overcome through the development of a progerin-specific antibody. Using this antibody, the progerin-positive fibroblasts were seen to increase in elderly skin [62]. Both prelamin A and progerin were also observed to accumulate in human vascular aging [63]. In a more recent study, a link between telomere dysfunction and progerin accumulation was established during normal aging. It was suggested that telomere shortening increased the production of progerin in normal cells and progerin and telomere dysfunction collaborate to trigger cellular senescence during normal aging [64].

A fundamental manner in which epigenetic misregulation in the form of DNA methylation and histone modification alterations can contribute to aging is by altering gene expression patterns. Since this has been the topic of discussion elsewhere [72], here we consider specific examples where epigenetic modifiers deregulated in aging may be involved in DNA repair. This point-of-view is especially important since the loss of DNA repair is a hallmark of aging and it is possible that epigenetic misregulation can contribute to aging by disrupting genome maintenance (summarized in Table 1).

Although it is known that the loss of genomic integrity is an important hallmark of aging, the molecular mechanisms are only beginning to be understood. Through the use of Zmpste24-knock mice as a model to understand premature aging, interesting insights have been obtained on how genomic integrity can be regulated by epigenetic mechanisms such as histone acetylation. H4K16 hypoacetylation affects global chromatin structure to impair DNA damage recognition and repair and this, in turn contributes to genomic instability during aging. Recent findings have indicated that the study of Zmpste24-null mice may have broader relevance in understanding physiological aging. Therefore, it would be interesting to find out if histone acetylation levels can also affect genomic integrity during normal aging. Given the inherent reversibility of epigenetic modifications, it would then be possible to manipulate histone acetylation levels and relax chromosome structure to promote DNA repair during normal aging and restrain some age-associated pathologies.

 

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